Hepatitis E virus RNA in commercially available porcine livers in The Netherlands

Hepatitis E virus (HEV) infections caused by genotype 3 are increasingly observed in industrialized countries, without a distinct source. High similarity between human and swine strains of HEV strongly suggest possible zoonotic transmission. It was reported previously that in 55% of Dutch pig farms HEV-excreting fattening pigs were present. In the current study, presence of HEV RNA in commercially available porcine livers was shown. We examined 62 commercially available porcine livers for HEV contamination. Before examination of livers, the most sensitive combination of tissue disruption and RNA-extraction was chosen from four disruption and seven RNA-extraction methods. Four of 62 livers were shown to be positive for HEV RNA by RT-PCR and Southern blot hybridization, and three sequences were obtained. Phylogenetic analysis showed clustering of the sequences with previously published Dutch HEV genotype 3 sequences from humans and swine. To study infectivity of possible virus, three pigs were intravenously inoculated with suspensions from commercially available HEV positive livers. Two other pigs served as high-dose or low-dose controls. The low-dose control received a comparable viral count as animals receiving inocula from commercially available livers, the high dose control received a viral count that was known to generate infection. Faecal shedding of HEV was observed in the high-dose control, indicating that the control virus was infectious. No faecal shedding of HEV was observed for the low-dose control and the three pigs that were administered the commercially available livers extracts. In conclusion, HEV RNA was found in commercially available porcine livers. inoculation of susceptible pigs with extracts from HEV-positive livers did not lead to infection, but this may be a dose-dependent effect. The risk for consumers should be investigated further

Comparison of methods for detection and typing of viruses in food : Hepatitis A virus and rotavirus

Voedseloverdraagbare virussen kunnen vooral worden overgedragen via voedsel dat niet of nauwelijks is verhit. Voorbeelden zijn schelpdieren, verse groente en fruit en kant-en-klare producten, zoals belegde broodjes en salades. Om de bron van een voedselinfectie te kunnen achterhalen wordt onderzocht of op het voedsel en in de patiënt hetzelfde virus aanwezig is. Tot nu toe werd hiervoor bij de patiënt een ander stukje van het genetisch materiaal van het virus aangetoond dan bij het virus dat aanwezig is op voedsel of in water. Door deze werkwijze was het niet mogelijk om de aangetroffen virussen te vergelijken, waardoor de besmettingsbron niet kon worden vastgesteld. Uit onderzoek van het RIVM blijkt nu dat zowel voor hepatitis A-virus als voor rotavirus één stukje genetisch materiaal kan worden aangetoond in de drie soorten monsters. Dit maakt het eenvoudiger om de bronnen van voedselgerelateerde virusinfecties te achterhalen. Voor het onderzoek zijn de verschillende methoden vergeleken die momenteel worden gebruikt om de bronnen van voedseloverdraagbare virusinfecties op te sporen. Virusonderzoek wordt vaak gedaan met de moleculaire techniek PCR (polymerase chain reaction), waarmee een deel van het genetisch materiaal van een virus wordt aangetoond. Er zijn verschillende PCR's beschikbaar waarmee verschillende delen van het genetisch materiaal worden gedetecteerd. Vaak wordt eerst aangetoond óf en welk virus aanwezig is (detectie). Vervolgens wordt bepaald om welk type virus het gaat (typering). Voor het onderliggende onderzoek zijn van twee virussen, hepatitis A-virus en rotavirus, meerdere detectie- en typerings-PCR's vergeleken. Voor beide virussen is bepaald welk stukje genetisch materiaal het best met PCR kan worden aangetoond om het in de mens, op voedsel en in water te kunnen detecteren. Daarnaast is bekeken welk stukje genetisch materiaal het meest geschikt is om het type virus vast te stellen (typerings-PCR). Voor beide virussen bleek dat de detectie-PCR kleinere hoeveelheden virus kan aantonen dan de typerings-PCR. Hierdoor is het mogelijk dat bij een 'lage' besmetting van het voedsel wel kan worden aangetoond dat een bepaald virus aanwezig is, maar niet kan worden bepaald om welk type virus het gaat. Dit betekent dat mogelijk niet zeker kan worden gesteld of het virus in de patiënt identiek is aan het virus op het voedsel. Dan blijft het onduidelijk of het verdachte voedsel ook daadwerkelijk de bron is. Aanbevolen wordt de methoden om virussen in voedsel aan te tonen en te typeren, te verbeteren.Foodborne viruses can be transmitted through the consumption of food that has had little or no treatment, such as shellfish, fresh produce and ready-to-eat food products. To be able to trace the source of a foodborne virus infection, viruses present in the food and in the patient have to be compared. Currently, a different part of the viral genome is detected in patients than in food and water samples, indicating that viruses cannot be compared and the source not established. Research by the National Institute for Public Health and the Environment (RIVM) has now determined which part of the viral genome of hepatitis A virus and rotavirus can be detected in all three sample types. This will facilitate the tracing of the source of foodborne virus infections. Several methods that are currently used for the tracing of viruses involved in foodborne infections were compared. Virus research is often done by the molecular technique PCR (polymerase chain reaction), which detects part of the genetic material of the virus. Several PCRs are available that target different parts of the viral genome. Generally, the presence of a virus in a suspected food item is determined by a detection PCR, after which the identity of the virus is established by a typing PCR. In the present study, several detection and typing PCRs specific for hepatitis A virus and rotavirus were compared. For both viruses it was determined which part of the viral genome was most efficiently detected in patients, food and water samples. Furthermore, the study showed which part of the viral genome was most suitable for typing the virus in the three different sample types. For both viruses, the detection PCR appeared to be more sensitive than the typing PCR, indicating that at low levels of contamination viruses may be detected, but cannot be typed. This means that viruses detected in the patient and in a suspected food product cannot always be conclusively determined as identical. If this is the case, then it remains unclear whether the patient contracted the infection through consumption of the suspected food product. It is recommended to optimize virus detection and typing methods in food.Nederlandse Voedsel- en Warenautoritei

Harmonization of virus diagnostics in human, food and environmental samples Inventory of methods used by VWA and RIVM

Bronopsporing van voedsel- en wateroverdraagbare virusinfecties is mogelijk met de methoden die gebruikt worden voor het aantonen van norovirus, hepatitis E virus en adenovirus in bron en patient, zodat maatregelen genomen kunnen worden om verdere ziektegevallen te voorkomen.VW

Hepatitis E Virus RNA in Commercial Porcine Livers in The Netherlands

Human hepatitis E virus (HEV) infections by genotype 3 strains in industrialized countries are hypothesized to be caused by pigs. To examine this hypothesis, the potential health risks of transmission routes should be examined. Possible foodborne transmission was studied by quantifying the presence and infectivity of HEV in commercial porcine livers in The Netherlands. A comparison of four tissue disruption and seven RNA extraction methods revealed that mechanical disruption followed by silica-based RNA extraction gave the highest RNA yields and was therefore employed on commercial porcine livers. Four (6.5%) of 62 porcine livers were HEV RNA positive by reverse transcriptase PCR and Southern blot hybridization. Each positive liver was estimated to contain ~65 PCR-detectable units per g. Sequences were obtained for three of four positive livers and classified as HEV genotype 3. Ninety-three percent similarity to Dutch human HEV sequences and 97% similarity to Dutch swine HEV sequences were observed. To determine whether positive livers contained infectious HEV particles, extracts from livers with known HEV RNA sequences were inoculated intravenously in pigs. Two control pigs were included: one was inoculated with a high dose known to result in infection (104 PCR-detectable units of HEV RNA), and the other was inoculated with a lower concentration of virus that equaled the concentration of PCR-detectable units in commercial livers (~20 PCR-detectable units). Infection was observed in the high-dose control, but not in other pigs, suggesting a dose-dependent response in pigs. Hence, the implications of HEV RNA in commercial porcine livers in The Netherlands are unknown. However, HEV RNA is present in commercial porcine livers, and sufficient heating of porcine livers before consumption as precautionary measure is recommended

Seroprevalence and molecular detection of hepatitis E virus in wild boar and red deer in The Netherlands

To date, sources of hepatitis E virus (HEV) in the Netherlands, including swine and wild boar, have been identified, but no direct attribution to Dutch hepatitis E cases have been demonstrated. Other animal sources may exist. To identify these species, HEV RNA detection by RT-PCR is required, but complicated. A preselection based on serology may be useful. Therefore, wildlife species were studied by serology and molecular methods. Using a species-independent double-antigen sandwich ELISA, HEV-specific antibodies were detected in sera from 12% of 1029 wild boar (Sus scrofa scrofa), in 5% of 38 red deer (Cervus elaphus) and in none of 8 studied roe deer (Capreolus capreolus). Differences in background signals were observed between species and accounted for by fitting finite mixture distributions. HEV RNA was detected in 8% of 106 wild boars, in 15% of 39 red deer and in none of 8 roe deer. In conclusion, HEV was shown to be present in European red deer for the first time. This preselection based on species-independent serological assays may be beneficial to identify new potential animal reservoirs of HEV. The consumption of Dutch undercooked wild boar and red deer meat may lead to human exposure to HE

Hepatitis E virus RNA in commercially available porcine livers in The Netherlands

Hepatitis E virus (HEV) infections caused by genotype 3 are increasingly observed in industrialized countries, without a distinct source. High similarity between human and swine strains of HEV strongly suggest possible zoonotic transmission. It was reported previously that in 55% of Dutch pig farms HEV-excreting fattening pigs were present. In the current study, presence of HEV RNA in commercially available porcine livers was shown. We examined 62 commercially available porcine livers for HEV contamination. Before examination of livers, the most sensitive combination of tissue disruption and RNA-extraction was chosen from four disruption and seven RNA-extraction methods. Four of 62 livers were shown to be positive for HEV RNA by RT-PCR and Southern blot hybridization, and three sequences were obtained. Phylogenetic analysis showed clustering of the sequences with previously published Dutch HEV genotype 3 sequences from humans and swine. To study infectivity of possible virus, three pigs were intravenously inoculated with suspensions from commercially available HEV positive livers. Two other pigs served as high-dose or low-dose controls. The low-dose control received a comparable viral count as animals receiving inocula from commercially available livers, the high dose control received a viral count that was known to generate infection. Faecal shedding of HEV was observed in the high-dose control, indicating that the control virus was infectious. No faecal shedding of HEV was observed for the low-dose control and the three pigs that were administered the commercially available livers extracts. In conclusion, HEV RNA was found in commercially available porcine livers. inoculation of susceptible pigs with extracts from HEV-positive livers did not lead to infection, but this may be a dose-dependent effect. The risk for consumers should be investigated further.</p

Development and Application of a Tool To Assess Laboratory Hygiene in Contained-Use Facilities▿

To gain information on laboratory hygiene in contained-use laboratories, a method was developed to study the presence of microorganisms on laboratory equipment. Focusing detection on genetically modified organisms (GMOs) containing the universal M13 primer binding sites enabled the detection of a broad range of GMOs using a single PCR. Swabbing surfaces in three different contained-use laboratories led to detection of M13-containing PCR products in 26 out of 34 swabs. Most sequences (up to five per sample) were detected in swabs from the centrifuge and sink, followed by swabs taken from the bin and incubator (up to four sequences per sample). The obtained sequences varied in length from 171 nucleotides (nt) to 878 nt. In most cases, sequences were only partially similar to sequences published in GenBank. The lengths of the regions with high similarity varied from 94 nt to 795 nt, and these similarities ranged from 81% to 100%. Similarities with more than one sequence were commonly found, complicating the identification of detected sequences. Nonetheless, 84% of the detected sequences were actually handled in the laboratory at the time of sampling. This demonstrates that the method may be used as a quality control tool to assess the efficacy of decontamination and cleaning of commonly used surfaces, such as laboratory benches, freezer doors, and centrifuge rotors, without prior knowledge of the identity or characteristics of the GMOs

Year-round screening of noncommercial and commercial oysters for the presence of human pathogenic viruses

Consumption of virus-contaminated shellfish has caused numerous outbreaks of gastroenteritis and hepatitis worldwide. In The Netherlands, oysters are cultured and imported both for consumption and export; therefore, the presence of noroviruses, rotaviruses, astroviruses, hepatitis A viruses, and enteroviruses was determined in 64 commercial and noncommercial oyster samples. Oysters were collected monthly for 13 months from four different harvesting areas in the Oosterschelde Delta. Oyster samples were classified by determining Escherichia coli levels according to the standards set by the Councils Directive (91/492/EEC). Two of 36 commercial and 2 of 28 noncommercial oyster samples were B-classified and therefore not ready for consumption. All other oyster samples were A-classified. For the detection of viral RNA, 150 mg of hepatopancreatic tissue was subjected to the Qiagen RNeasy Mini Kit, followed by reverse transcriptase (RT)¿PCR and Southern blot hybridization. Enterovirus RNA was detected in 14 of 64 oyster samples, of which 4 were from noncommercial oyster harvesting areas and 10 were from commercial harvesting areas. None of the other human pathogenic viruses were detected. The levels of somatic coliphages and F-specific phages were also determined in all 64 oyster samples, with some samples containing high phage levels (>50 PFU/g of hepatopancreatic tissue), but with most samples containing low phage levels

Prevalentie van antibioticaresistente bacteriën in Maas, Rijn en Nieuwe Maas

In de grote Nederlandse rivieren de Maas, de Rijn en de Nieuwe Maas komen bacteriën voor waarvan hoge percentages resistent zijn tegen een of meer soorten antibiotica. Dit blijkt uit verkennend onderzoek van het RIVM. Blootstelling via oppervlaktewater: Als mensen aan verontreinigd oppervlaktewater blootgesteld worden, kunnen zij antibioticaresistente bacteriën binnenkrijgen. Dit kan bijvoorbeeld via recreatiewater of via water dat gebruikt worden om gewassen te besproeien. Dergelijk contact kan risico's voor de volksgezondheid met zich meebrengen, omdat deze antibiotica nodig zijn om infecties te behandelen. Antibioticaresistente bacteriën kunnen op meerdere manieren in oppervlaktewater terechtkomen, bijvoorbeeld doordat mest van dieren die met antibiotica zijn behandeld, afspoelt naar het oppervlaktewater. Een andere oorzaak kan zijn dat gedeeltelijk gezuiverd of ongezuiverd afvalwater in oppervlaktewater wordt geloosd, bijvoorbeeld afkomstig van ziekenhuizen waar mensen zijn behandeld met antibiotica. In totaal waren gemiddeld eenderde tot de helft van alle Escherichia coli en van de intesintale enterococcen resistent tegen een of meer soorten antibiotica. In sommige van de monsters werden antibioticaresistente stammen van Staphylococcus aureus, Campylobacter en Salmonella aangetoond. De meeste van deze bacteriën zijn darmbacteriën; Staphylococcus aureus komt vooral voor op de huid, en in de neus en keel van mensen. Diverse risico's: De risico's kunnen zich op verschillende manieren manifesteren. Op de eerste plaats kunnen mensen die aan antibioticaresistente bacteriën worden blootgesteld, daarvan ziek worden en vervolgens problemen krijgen bij de behandeling ervan. Daarnaast is het mogelijk dat mensen die worden blootgesteld aan de resistente bacteriën zelf niet ziek worden, maar deze overdragen aan mensen met verminderde weerstand, zoals ziekenhuispatiënten en ouderen. Deze groep mensen kan vervolgens wel ziek worden door deze bacteriën. Ten slotte is er het risico dat onschadelijke antibioticaresistente bacteriën zich in de darmen nestelen en daar genen die resistentie veroorzaken doorgeven aan andere, ziekteverwekkende bacteriën. Nader onderzoek naar volksgezondheidsrisico's: Onderzoek naar de mate waarin antibioticaresistente bacteriën in oppervlaktwater voorkomen is van belang om te kunnen inschatten in hoeverre mensen via het milieu worden blootgesteld aan deze bacteriën. Nader RIVMonderzoek zal hierop gericht zijn, en wat dit betekent voor de volksgezondheid.A high percentage of bacteria in large Dutch rivers is resistant to one or more antibiotics. This was demonstrated in an exploratory study performed at the RIVM. Exposure through surface water: If people come into contact with contaminated surface water, they risk exposure to bacteria that are resistant to one or more antibiotics. This may occur for instance during recreation in surface water or through water used for irrigation of crops. Because antibiotics are needed for treatment of infections, such contact may involve public health risks. Antibiotic resistant bacteria can end up in surface water by different routes, for example through run off of manure from animals treated with antibiotics to surface water. Another source may be the discharge of partially treated or untreated waste water, for instance derived from hospitals where people are treated with antibiotics. Overall, one third to half of all Escherichia coli and intestinal enterococci were resistant to one or more antibiotics. In some of the samples antibiotic resistant strains of Staphylococcus aureus, Campylobacter and Salmonella were detected. Most of these bacteria are gut bacteria; Staphylococcus aureus is predominantly present on people's skin and in their noses and throats. Diverse risks: The risks can manifest themselves in different ways. Firstly, after exposure to antibiotic resistant bacteria people may develop disease caused by these bacteria, which may therefore be hard to treat. Additionally, people may not become ill themselves, but transfer the resistant bacteria to people who are more vulnerable, such as hospital patients and the elderly. Subsequently, this category of people can develop disease. Finally, there is a risk that the antibiotic resistant bacteria establish themselves in the gut and transfer resistance genes to disease-causing bacteria if these are subsequently ingested Further research on public health risks: Data on the prevalence of antibiotic resistant bacteria in surface water are necessary to estimate the contribution of the environment to human exposure to these bacteria. Further RIVM research is focused on this type of exposure and the associated possible public health risks.VROM-Inspecti